These studies are designed to examine 44 human anti-V3 monoclonal antibodies (mAbs) with respect to how the immunoglobulin (Ig) variable heavy (VH) and variable light (VL) chain gene usage determines Ab cross-reactivity and function. The studies are based on previous findings in that human Abs against different pathogens exhibit preferential VH gene usage. In this application we focus on Abs to the V3 loop, a hypervariable region of the HIV-1 gp120 envelope glycoprotein, which may require very different structural features in the Abs that bind to it in order to accommodate its sequence heterogeneity. Human anti-V3 mAbs have the capacity to neutralize primary isolates and this suggests that Abs against V3 might constitute an important target for HIV vaccine. The most useful approach to study the role of Ig genes in targeting particular epitopes is the analysis of the VH and VL germline gene usage among mAbs. Therefore, in Aim 1 we will evaluate the VH and VL gene usage for neutralizing anti-V3 mAbs and compare this with mAbs specific to the CD4-binding domain, the CD4i epitope, and gp41. Preliminary analysis of several human anti-V3 mAbs has revealed a preferential usage of the VH5-51 gene segment that is rarely used within the normal repertoire. Complete analysis of the 44 anti-V3 mAbs will be used to determine the extent of the biased gene usage. In Aim 2, we will address the question of whether there is a correlation between VH and/or VL gene usage and cross-neutralizing activity of anti-V3 mAbs. Pilot experiments support the hypothesis that preferentially used VH genes may encode mAbs displaying broader cross-reactivity and more efficient neutralization. In Aim 3, we will examine the relationship between gene usage and mAb activity by crystallography, analyzing the antigen-interacting residues in VH and/or VL and sequence analysis of the germline genes encoding these mAbs. We hypothesize that the germline genes preferentially used by the V3 mAbs may encode residues which interact with the antigen and remain intact in the CDRs of the antibody. The results impact on understanding the immunogenetics of Ab production which could have critical implications for HIV vaccine development. NARRATIVE: In this proposal, we will study immunoglobulin gene usage for the human neutralizing antibodies against the V3 region of HIV-1. Our preliminary results suggest that there is a preferential use of one particular immunoglobulin gene segment for making anti-V3 antibodies and that the resultant antibodies are very efficient at neutralizing viruses from different HIV-1 subtypes. Identifying the immunoglobulin genes that encode the most potent neutralizing antibodies will be essential when designing an effective HIV vaccine with capability of inducing protective antibodies.